Parkinson's disease (PD) is characterized by aggregation of alpha-synuclein (a-synuclein) into Lewy bodies and Lewy neurites, and a progressive loss of specific neuronal populations. In particular, ventral midbrain (VM) dopamine (DA) neurons of the substantia nigra (SN) preferentially degenerate in PD, while neighboring ventral tegmental area (VTA) DA neurons are relatively spared. Our previous studies indicate that multiple hits, consisting of high cytoplasmic Ca2+, elevated cytosolic DA (DAcyt) and a-synuclein expression are required to evoke selective death of SN dopaminergic neurons and show that interference with any of these three factors rescues the neurons. Here we propose to examine the molecular mechanisms responsible for the toxic interaction between DAcyt, a-synuclein and chaperone-mediated autophagy (CMA), a pathway for selective lysosomal degradation of cytosolic proteins. In particular, we will investigate the possibility that multiple feedback loops between a-synuclein, DA and CMA exacerbate cellular stress and lead to selective degeneration of SN neurons. Previous studies in vitro and on non-neuronal cells suggest that a-synuclein overexpression induces a leakage of neurotransmitter from synaptic vesicles, disrupts stimulation-dependent neurotransmitter release and elevates DAcyt. In Aim I, we will determine the effect of a- synuclein overexpression or mutation on neuronal DAcyt and quantal DA release. Additionally, we will study the effect of a-synuclein and DAcyt on mitochondrial redox status and neurotoxicity and will assess the specificity of these effects for neurons from SN and locus coeruleus (LC). Next, it has been shown that pathogenic and DA- modified a-synucleins block CMA, which may further increase the level of a-synuclein by preventing its degradation through this pathway. In Aim II, we will study the synergistic effect of a-synuclein and DAcyt on cellular stress and CMA activity. First, we will determine whether CMA blockade requires a direct interaction between DAcyt and a-synuclein. Second, we will investigate if CMA blockade increases a-synuclein levels, promotes its oligomerization and increases DAcyt. Third, we will study whether enhanced CMA activity rescues cells from DA-a-synuclein-induced toxicity. This project will provide important information about the pathways that lead to differential susceptibility of neuronal populations to stress, which is important for the development of treatments that prevent the death of catecholaminergic neurons in PD.